Drugs that act intracellularly generally enter cells by diffusion. Most drugs are small molecules because they have the ability to diffuse across tubulovesicular structures or organelle membranes to reach their site of action. To increase the bioavailability of a drug, often small molecules must be modified and/or formulated for greater solubility and/or permeability, depending on route of administration. Even small diffusible drugs may not be efficacious at their site of action. For example, multidrug resistance (MDR) may be present, which results in active efflux of drugs that enter cells with MDR. MDR often occurs in cancer cells.
In contrast to small molecules, high molecular weight compounds and polymer drugs, such as polynucleotides, polypeptides, and other macromolecules have little to no ability to diffuse across membranes. High molecular weight material is generally internalized by endocytosis. The addition of affinity binding partners to high molecular weight material can direct the high molecular weight compound to specific cells, and thereby result in increased selective uptake. However, once endocytosed, the material still remains separated from the cellular cytoplasm by a biological membrane.
Furthermore, endocytosed material is often delivered to the lysosome, where material sensitive to lysosomal enzymes is quickly degraded if steps are not taken to protect its breakdown or to facilitate escape from the lysosome. Delivery of high molecular weight compounds to their site of action at effective levels is a problem. It is therefore desirable to improve delivery to a desired subcellular compartment.
An aspect of the invention is to provide novel tubular vesicular structure localization signals by modifying one or more proteins capable of locating to the tubulo-vesicular structure of polarized cells by truncation or by amino acid substitution. Truncations, amino acid substitutions, and other modifications of known tubulo-vesicular structure-locating proteins are made to minimize endogenous biological activities other than localization. In general, the invention relates to cellular localization signals. More specifically, the invention relates to tubulovesicular structure localization signals in polarized cells. In polarized cells, such as kidney epithelial cells, which contain membranes, localization signals are selective for one of these tubulo-vesicular structure locations.
The polarized cell tubulo-vesicular structure localization signals are monomeric units that can be used separately or together for the purpose of targeting a polypeptide or other molecule of interest to a desired tubulo-vesicular structure location. Monomeric units used together exploit cooperation and synergism among individual signals in order to enhance strength and/or performance of individual signals. For example, some localization signals function at the N-terminus of a polypeptide of interest, and other localization signals function at the C-terminus of a polypeptide of interest. The signals of this invention encompass localization signals placed toward the N-terminus or C-terminus, or both the N-terminus and C-terminus of a polypeptide or other molecule of interest.
The localization signals are utilized as research tools or are linked to therapeutics. Disclosed are methods of making and using polypeptides and modified polypeptides as signals to localize therapeutics, experimental compounds, peptides, proteins and/or other macromolecules to the tubulo-vesicular structure of polarized eukaryotic cells, such as kidney epithelial cells. The polypeptides of the invention optionally include linkage to reporters, epitopes and/or other experimental or therapeutic molecules. The invention also encompasses polynucleotides encoding the localization signals and vectors comprising these polynucleotides.